1. Field of the invention
The present invention relates to a novel host vector system. In particular, the present invention relates to novel host vector system in which hosts are Rhizopus niveus and related molds belonging to phycomycetes, and vectors are also derived from various phycomycetes species.
2. Description of the related art
A method for preparing useful proteins such a interferon, interleukin and others by genetically engineere bacteria has been established. However, problems are no occurring in the method using bacteria as hosts. Of example, the extracellular secretion of the proteins which is sometimes very necessary for the production of large amounts of target proteins is not very efficient in many bacteria Moreover, the intracellularly produced interferon or interleukin produced in E. coli has the wrong secondary structures which is different from that of the natural protein. As a result, the artificial protein is recognize. in a human body as an antigen. Thus denaturation an renaturation procedures of the artificial protein are required prior to the pharmaceutical use of the protein.
An, important advantage of a fungal genetic engineering system is its ability to produce glycosylated protein which is impossible with bacterial host vector systems. Many human and animal hormones and other physiologically important proteins are attached with oligosaccharide chains. These glycomoieties are sometimes necessary for the physiological functions of the human hormones as in the case of erthropoietin, and in many cases increase the stability of target proteins against the attack of proteinases in the culture medium, in the injected body fluids, ect.. Each fungi and yeasts produce different structures of glycomoieties, which gives different recognition signals to the human immunological systems and others. Therefore, the construction of various fungal genetic systems are important. The host vector systems using animal and human cells as hosts produce similar or correctly glycosylated proteins, however, their ability to produce of the target proteins are much lower than the fungal system.
Phycomycetes such as Rhizopus or Mucor are used frequently in fermentation technology. They are classified into Phycomycetes. Rhizopus has the extensive ability to secrete enzymes and proteins extracellularly For instance, the amount of glucoamylase secreted by Rhizopus niveus into the liquid culture medium is about 2 g/liter and that secreted with solid culture procedure it about 30 g/kg. Further, not only the primary structure but the secondary structure of the resulting protein is the same as that of a natural protein due to the extracellular secretion system through membrane. Therefore, it is expected that transformed molds will be used for the preparation of various kinds of commercially important enzymes and proteins. However, only a few transformation techniques are established for fungi and no transformation techniques for Rhizopus known. Under the circumstances, the present inventors successfully provided a transformation technique used for Rhizopus(see Japanese Patent Disclosure (KOKAI) (JP-A-) 2-53480). However, a vector suitable for producing an enzyme by using Rhizopus as a host has not been known.
An object of the present invention is to provide a host vector system suitable for the production of proteins and enzymes using. Rhizopus as a host Rhizopus has the enhanced ability to produce proteins and enzymes extracellularly and in the cell because of their large cell mass production